Chitosan exerts cytotoxic activity in vitro by inducing apoptosis and necrosis in mouse hepatoma and chinese hamster ovary cells

Abstract

The mechanisms responsible for the cytotoxic effects of chitosan on cancer cells are still unclear. The goal of this study was to evaluate the cytotoxic properties of chitosan films and solutions in vitro on mouse hepatoma MH-22A (tumor cells) and Chinese hamster ovary (CHO, non-cancerous) cells cultivated on films with different chitosan concentrations and cultivated on Petri dishes after preincubation with chitosan solutions. To measure plating efficiency (cloning efficiency), cells were plated at low densities (~300 cells per dish) on chitosan films and allowed to grow for 9 days. To see the colonies clearly, they were stained with crystal violet. To investigate cytotoxic activity of chitosan in cell suspensions, 10μl of the chitosan solution (10, 1, 0.1 and 0.01 gL-1)/acetic acid was added to 90μl of warm, fresh serum-free media such that there were duplicate tubes per dose including the untreated control. The cells were mixed gently by pipetting every 10 min during 20 min and 1h and then plated and allowed to grow at 37 oC and 5 % CO2 for 5–6 (CHO cells) or 9 days (MH-22A cells). To reveal the rupture of cell membrane (morphologic expression of necrosis) cells were stained with Trypan Blue solution after 40 and 100 min of chitosan treatment. Cells fully or partly did not attach to the surfaces of Petri dishes, coated with chitosan solutions with concentrations ranging between 10 and 2 gL-1 (p<0.05). In these dishes on the next day after seeding cell debris was observed on the top of culture media, that is chitosan films not always have surfaces with adhesive for mammalian cells properties. At lower concentrations (between 0.2 and 1 gL-1), cells formed both normal and sparse colonies. With chitosan concentrations ranging between 1 and 0.01 gL-1 cells had surviving fraction more than 70%. After the incubation of cells with chitosan solutions, morphologic features of necrosis and apoptosis were observed.[...]